High Sensitivity Tetraspan Tracking (HSTT) ELISA Kit
Research Use Only Not for Diagnostic Use
Description: EVix High Sensitivity Tetraspan Tracking (HSTT) Kit. This complete assay kit allows assessment of tetraspan antigen in an EV sample. EV are captured on individual wells coated with anti CD9,CD63 and CD81 monoclonal antibody. The tri-antibody coating maximizes capture of EVs expressing any or all of the three tetraspan antigens.
The high affinity and specificity of the capture provides sufficient capture in two hours of incubation. The detection antibody cocktail yeilds high sensitivty and allows quantiification of EV preparations based on antigen content. In contrast to NTA analysis which can be time consuming to run multiple samples, the HSTT assay can quantitate antigen content of an EV preparation in less than 4 hrs total. The assay is straightforward, sensitve and reproducible. A 2 hour capture is followed by a 1 hour detection and a 30 minute development process.
The HSTT assay includes an internal standard that yields a highly reproducible signal and can be used to calibrate EV preparations. This allows dilution or concentration of EV preparations to achieve equivalency for downstream investigation. The HSTT assay can be used to assess multiple EV samples from the same source or to compare EV preparations from different sources.
The HSTT assay kit provides 12 modular 8 well strips for flexibility in assay design. A single strip can be used if one has a small sample to evaluate, the 8 wells provides testing of a negative control, positive control, and EV sample/s to derive a HSTT value for the samples. The investigator can run repeat assays from a single HSTT assay kit or can use the entire 96 wells to screen a large number of samples or to capture dilution curves for multiple samples.
The HSTT assay kit comes complete with all reagents needed to obtain results. No other reagents are needed to obtain values for EV samples. The HSTT assay is the answer to quantifying EV preparations. Simple, rapid, highly sensitive and reproducible. Start measuring your EV preparations based on non destructive antigen capture and recognition. Track your purification strategies, calibrate preparations, compare recovery rates, and more.
HSTT Values for Tracking Tetraspan Content
Use HSTT Assay to monitor Tetraspan antigen content in your EV preparations. High sensitivity and specificity capture and detect with anti tetraspan monoclonal antibody creates values for EV preparations that can be used to equilibrate or normalize EV preparations from the same preparation method.
HSTT values can be used to guide dilution and concentration of preparations for down stream analysis. These values allow for normalizing samples the Multi-Tope and Single-Tope assays. Samples diluted to give equivalent HSTT values can be run in Multi-Tope and Single-Tope assays and compared for antigen distribution patterns.
Complement NTA analysis. Once your purification process is locked in, HSTT can substitute for the laborious NTA analysis to quantify EV preparations. Normlize the NTA values to HSTT readings and you can substitute HSTT to follow EV containing antigen amounts in your experiments.
Sensitive and Reproducible
Replicate measurements of an EV preparation shows reproducibility of HSTT values. Three different assays ran on the same sample. Values above 3.5 AU are close to saturation. EV preparation diluted in wash buffer by serial dilution. EV preparation from SW 620 culture supernatant diafiltered against 300,000 MWCO concentrator.
Quantify EV Preparations
Linearity of assay to antigen amounts. Three different assays of the same EV preparation demonstrate sensitivity and linearity of assay values to increasing amounts of antigen. Values above 3.5 AU are close to saturation. EV preparation diluted in wash buffer by serial dilution. EV preparation from SW 620 culture supernatant diafiltered against 300,000 MWCO concentrator. A 1 to 10 dilution of supernatant yielded near maximum assay values.
Normalize Different EV Preparations
EV Preparations from 10 different biological sources were assayed at 4 dilutions to compare HSTT values. Each preparation yielded different HSTT dilution curves. Samples included normal urine, MLR supernatants, and cell line supernatants. All samples were dia-filtered against a 300,000 MWCO membrane.
Dilute or Concentrate EV preparations to equivalent HSTT values
Dilution of EV Preparations from 10 different biological sources reveal unique HSTT dilution values for each preparation. Dilution from concentrate for all samples are shown. Samples included normal urine, MLR supernatants, and cell line supernatants. All samples were dia-filtered against a 300,000 MWCO membrane.
Nomal human serum was ran on a S300 HR Sephacryl chromatography column. 0.5 ml of serum was loaded onto column and 0.5 ml fractions collected. The 260 and 280 absorbance values for the fractions are shown on the left vertical axis. HSTT values of the individual fractions are shown on the right vertical axis. Note robust HSTT signals in the exclusion/void volume but not in the inclusion volume. IgM also came out in void volume.
HSTT signals were readily detected even when serum was diluted several fold due to fraction collection.
Rapid and Sensitive
Assay multiple samples of EVs simultaneously in a single assay. The assay requires only 2 hr to capture and 1 hr to detect.
Assay multiple samples with high sensitivity and reproducible results
Detection of EVs in straight serum samples, allowing for chromatography separation of EVs from other serum components.
Multiple samples can be run simultaneously to assess multiple populations and dilutions. These values can be used to prepare equivalent preparations of EVs in place of NTA measurements.
Standardize EV preparations
Dilute and equilibrate EV populations to allow comparisons between samples.
Qualify EV preparations
The HSTT assay allows equivalent EV antigen content to be assayed in downstream Multi Tope and Single Tope assays.
A: EVix Capture Plate: (12 x 8 well strips) and frame.
B: 10X Sample and Wash Buffer Concentrate: 2 bottles (10 ml each) to make 200 ml.
C: Biotinylated Primary Detection Antibody: 1 vial of 0.5 ml to make 10 ml.
D: Streptavidin Horse Radish Peroxidase SAHRP Conjugated: 1 vial of 0.5 ml to make 10 ml.
E: Chromogenic Solutions : 1 bottle (10 ml) of stabilized H2O2 , 1 vial (0.5 ml) of stabilized TMB chromogen. Caution!
F: Stop Solution: 1 bottle (10 ml) Acid Caution!
G: EVTetraspan Standard: 1 vial 0.5 ml.
If contents are unopened and stored at 4o C the kit is stable for 6 months.
Order HST-001. 96 wells in 8 well modular strips. All assay components included.
Catalog HSTT 001